Tumors of the Ewing's sarcoma family (ESFT) are solid, highly malignant neoplasms of the bone and soft tissues that most often affect children and adolescents, being the second most common bone malignancies among young adults. Current treatment includes a combined modality with chemotherapy and radiotherapy. However, there are two important problems to consider: a) although ESFT are generally responsive to treatment, the overall cure rate is low because these tumors are very aggressive and patients frequently present with metastatic disease; and b) exposure of young patients to high doses of chemotherapy and/or radiation is frequently associated with a variety of adverse health effects that in some cases do not develop until many years after treatment completion. Consequently, treatment strategies are needed to maximize curability while simultaneously minimizing adverse late effects for the patients. In preliminary studies, we directly targeted EWS/FLI-1, the transcription factor known to be responsible for the malignant properties of most ESFT, using molecular tools designed to block its activity, either alone or in combination with chemotherapeutic drugs and/or radiation. Although experiments in vitro and in mouse xenografts demonstrated that these tools efficiently delayed tumor growth, it became clear that simultaneous targeting of additional molecules that act downstream of EWS/FLI-1 would be necessary to maximize antitumor activity and to allow the use of lower therapeutic doses, thus minimizing negative late effects as much as possible. In this context, the main objective of this proposal is to exploit a novel EWS/FLI-1-driven pathway recently established in our laboratory (EWS/FLI-1-[Caveolin->1-Snail-E-cadherin]) to sensitize ESFT cells to therapy, using ionizing radiation (IR) as a model anti-neoplastic agent that does not suffer from the specificity- related problems that frequently complicate studies with chemotherapeutic drugs. Since the extent of IR related late effects in normal tissues surrounding the tumor are directly related to the IR dose, in pediatric cancers it is important to employ the lowest dose possible to cure the tumors. Using EWS cells as the ESFT prototype, our central hypothesis is that (a) targeting caveolin-1 (CAV1) itself and/or its interactions with signaling or regulatory proteins relevant for ESFT molecular pathobiology (such as phospholipase D2 and protein kinase C1) will render EWS cells more sensitive to IR, and (b) that this response may be further improved by simultaneously targeting components of other pathways also known to radiosensitize EWS cells, such as poly (ADP-ribose) polymerase (PARP). Because we already identified CAV1 as a direct transcriptional target of EWS/FLI-1 and a key determinant of the tumorigenicity and chemotherapeutic response of EWS cells, targeting CAV1 signaling may have a dual effect: enhancing killing of EWS cells by low-dose IR and down- regulating their neoplastic properties.